CaM kinase II is a major mediator of Ca2+ -linked signal transduction systems; it phosphorylates diverse substrates located in cytosolic, nuclear, cytoskeletal, and membrane compartments. How is response specificity achieved if any rise in Ca2+ activates all Ca2+ -dependen processes throughout the cell and if CaM kinase II responds by phosphorylating all its substrates? They have found temporal and spatial elements of signal transduction which may enable response specificity by CaM kinase II. Temporal regulation: Many cell stimuli subject the kinase to oscillations in the concentration of intracellular Ca2+. Although information is thought to be encoded in the stimulus frequency, no frequency decoder has previously been identified. They have now demonstrated that CaM kinase II is sensitive to the frequency of Ca2+ oscillations in vitro with the use of a pulse flow device that exposes immobilized kinase to Ca2+ pulses under precise kinetic control and which they can now exploit. They will examine the molecular basis for this biochemical behavior, defining the relative contribution of various steps in kinase activation and deactivation to the frequency response. They will use kinase constructs as probes of effective Ca2+/CaM concentration to test of whether cellular CaM is limiting and assess its contribution to the frequency response. They will then test whether CaM kinase is sensitive to the frequency of Ca2+ oscillations in situ using several cell lines with defined Ca2+ signaling pathways and intracellular targets of the kinase. Spatial regulation: They have found that CaM kinase isoforms are targeted to distinct cellular localization and that cell signals can have preferential regulation of the kinase based on its localization. They will explore our recent finding that targeting of a nuclear isoform of the kinase is regulated by Ca2+-dependent autophosphorylation as well as phosphorylation by other kinases. They will test the ability of various cell stimuli to spatially direc their signals by engineering cells with CaM kinase II targeted to distinct cellular localization as a spatial probe to cell signaling. Signal transductio pathways may both control activation of the kinase as well as its intracellula targeting and thereby regulate the specificity of cellular responses to stimulation.
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